ABSTRACT: A major goal of cancer immunotherapy has been to re-activate quiescent tumor-associated T-cells to enhance their detection and killing of cancer cells in tumor microenvironments. It is well established that with persistent activation of T- cells, in chronic inflammation and cancer, immune suppressive checkpoints exist that inhibit T- cell activation to limit collateral damage to host tissues. Building on this knowledge cancer therapies have been developed that block T-cell checkpoints using monoclonal antibodies. Checkpoint blocking strategies that have targeted the inhibition of two T-cell checkpoints, PD-1 and CTLA-4, have been curative for some cancers. However, a majority of patients either do not respond or the responses are not durable. A likely reason for this is the presence of other immune regulatory systems that suppress T-cell function in tumors, including T regulatory cells and myeloid derived suppressor cells that perhaps must also be eliminated. Recently there has been a growing awareness that nano-sized extracellular vesicles present in tumors are able to arrest T-cell function. We have isolated micro-vesicles called exosomes (EX) from human tumors that bind to and internalize into T-cells resulting in a rapid and reversible blockade in the activation potential of these cells. The immunosuppressive EX represents a new T- cell checkpoint that results in an arrest of the activation the T-cell signaling cascade. The suppression of T-cells has been causally linked to phosphatidylserine (PS) expressed on the surface of the EX. Several non-toxic water soluble organic molecules that bind PS have been synthesized by our collaborators at MTTI, and have been screened and shown by us at IMT LLC, to block/reverse the immune suppressive activity of tumor-associated EX. One of the PS binding molecules, Zn- T-DPA, significantly inhibits the T-cell immune suppression of the tumor-associated EX in vitro. . In Aim 1 the pharmacokinetics (PK), the bio-availability and toxicity of Zn-T-DPA, will be addressed in globally immune deficient NSG naive mice, and in these mice bearing human ovarian tumor xenografts. In Aim 2 we will determine the pharmacodynamics (PD) of this molecule in vivo. We predict, and will test here, that treatment in vivo with Zn-T-DPA (at a dose determined in Aim 1) of NSG mice bearing human ovarian tumor xenografts will block or reverse the T-cell suppression by the tumor-associated exosomes, re-activate patients? tumor- associated T-cells, and enhance tumor killing in the tumor microenvironment. With our novel xenograft model (that includes tumor stroma, the tumor-associated T-cells, and exosomes) we are able to quantify several matrices including changes in tumor cell number, serum levels of human cytokines, and in the number and activation potential of the tumor- associated T-cells. Our in vivo studies in Phase 1 of this application are expected to provide a rationale and underpinning for a Phase II application to study the PK, PD and efficacy of the Zn-T-DPA in combination with currently used checkpoint inhibition therapies, and provide for a scale up development of the Zn-T-DPA for a Phase I clinical trial.